Staphylococci are mainly found on the skin or in the nose. These bacteria are typically friendly, causing no harm to healthy individuals or resulting in only minor issues that can go away on their own. However, under certain circumstances, staphylococcal bacteria could invade the bloodstream, affect the entire body, and lead to life-threatening problems like septic shock. In addition, antibiotic-resistant Staphylococcus is another issue because of its difficulty in the treatment of infections, such as the notorious methicillin-resistant Staphylococcus aureus (MRSA) which is resistant to most of the currently known antibiotics. Therefore, rapid and accurate diagnosis of Staphylococcus and characterization of the antibiotic resistance profiles are essential in clinical settings for efficient prevention, control, and treatment of the bacteria. This chapter highlights recent advances in the diagnosis of Staphylococci in clinical settings with a focus on the advanced technique of surface-enhanced Raman spectroscopy (SERS), which will provide a framework for the real-world applications of novel diagnostic techniques in medical laboratories via bench-top instruments and at the bedside through point-of-care devices.